N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 3, 2016 - November 24, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]

EC Number:

240-354-5

EC Name:

N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]

Cas Number:

16230-35-6

Molecular formula:

C19H24N2O3Si

IUPAC Name:

N,N'-[ethoxy(methyl)silanediyl]bis(N-methylbenzamide)

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature.

In vitro test system

Test system:

human skin model

Remarks:

SkinEthic™ RHE model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Details on animal used as source of test system:

The SkinEthic™ RHE model has been validated for irritation testing (Validation study based on the original ECVAM Performance standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439), therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied as supplied

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE model.
- Tissue batch number(s): 16-RHE-122.
- Delivery date: 22 November 2016.
- Expiration date: 28 November 2016.
- Date of initiation of testing: 22 November 2016.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature.
- Temperature of post-treatment incubation (if applicable): 37ºC.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 ml of DPBS.
- Observable damage in the tissue due to washing: no.
- Modifications to validated SOP: no.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL.
- Incubation time: 3 hours at 37ºC, 5% CO2.
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek).
- Wavelength: 570 nm.
- Linear OD range of spectrophotometer: The linearity range of the optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.2 (CV=4.6%) (Specification OD>0.7).
- Barrier function: 5.2 h (Specification 4 h- Morphology: 6 cell layers, absence of significant histological abnormalities, well differenciated epidermis (Specification >4).
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is less or equal than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours and 15 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

ca. 58.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

1.0%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: A yellow solution was observed after 3 hours of incubation between 36.4ºC and 37.8ºC, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT: In isopropanol: a colorless solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank substracted) was 0.001 which is less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore, the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, a full demonstration of proficiency was performed with Episkin-SM model, plus a reduced validation with SkinEthic™ RHE model. Adequate results were obtained for the evaluated chemicals. summary of proficiency chemicals tested according to OECD 439 criteria included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the standard deviation of the negative control group was 21.3%, instead of ≤18 as initially scheduled. This is due to a mean optical density (OD) vallue of the replicate. No. 3 treated with the negative control which is slightly above the maximal OD value of 1.5. The higher value obtained in the negative control group could lead to an over-classification of the test item. Considering the results obtained (the cell viability of the three treated epidermises is under 50% with a standard deviation of 4.5%), this deviation is considered as without impact on the conclusion of the study (the test item is not irritant for the skin).
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: yes.

Any other information on results incl. tables

The results were expressed as a viability percentage compared with the negative control:

 

Viability %= OD_{test item}/ OD_{negative control}x 100

 

The OD values obtained for each test simple were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%.

 

Table 1. Individual and average values of OD after 42 minutes exposure

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD	Conclusion
Negative control	1	0.943	1.019	1.309	77.8	100.0	21.3
		1.039
		1.075
	2	1.308	1.334		101.9
		1.352
		1.343
	3	1.512	1.575		120.3
		1.557
		1.657
Positive control	1	0.012	0.013	0.013	1.0	1.0	0.1	Irritant
		0.013
		0.013
	2	0.011	0.011		0.8
		0.012
		0.011
	3	0.013	0.014		1.1
		0.014
		0.014
Test item PH-16/0505	1	0.799	0.836	0.768	63.8	58.7	4.5	Non irritant
		0.756
		0.730
	2	0.738	0.741		56.6
		0.756
		0.730
	3	0.738	0.728		55.6
		0.761
		0.686

# mean of 3 values (triplicate of the same extract)

OD: optival density

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

 

Acceptability criteria:

-         Negative control: OD values of the 3 replicates in the range≥ 0.8 and ≤ 3.0 for SkinEthic RHE model; and SD value of the % viability ≤18%. As the optical density was measured after a 1:2 dilution of the extracts, OD values of the 3 replicates should be in the range≥0.4 and≤1.5.

-         Positive control: classified as irritant, and SD value of the % viability≤18%.

-         Test item: same classification for the 3 replicates, and SD value of the % viability≤18%.

 

Notes:

-         If the viability obtained for the test item is greater tan 50%, the test item has to be considered as non irritant.

-         If the viability obtained for the test item is less tan or equal to 50%, the test item has to be considered as irritant.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corrected percent viability of the treated tissues was 58.7%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to skin.

Executive summary:

The evaluation of the possible irritating effects of the test item has been tested after topical application on in vitro human reconstructed epidermis ( SkinEthic™ RHE model) in accordance with OECD 439, following GLP. The test item was applied, as supplied, at a dose of 16 µL to 3 living skin models during 42 minutes at room temperature, followed by a rinse with 25 mL of DPBS and a 41 h and 15 min post-incubation period at 37ºC, 5% CO2. Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan sant that was quantitatively measured after extraction from tissues. Negative and positive controls were run in parallel. The mean corrected percent viability of the treated tissues was 58.7%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). No other effects were observed. In accordance with the Regulation EC No. 1272/2008, the test item has to be considered as non-irritant to skin, corresponding UN GHS No category.